The regenerative medicine which involves transplanting a tissue produced from stem cells into a lesion and realizes regeneration of injured tissues and organs and restoration of functions thereof has been attracting attention in recent years. It is known that transplantation of the cell sheet which is a living body imitation tissue especially has higher recovery effects compared with a cell suspension in which cells exists singly, so that cell sheets have been increasingly applied clinically. For example, human epidermal cell sheets have been commercially manufactured and clinically to treat serious burns and other problems. One of the objects currently left to be solved regarding this cell sheet is the establishment of a non-invasive evaluation method of the cells state of the cell sheet.
FIG. 2 shows the culture steps until a cell sheet is normally produced. FIG. 2 shows the structure of cells observed laterally, when a culture surface 201 is in the direction of the width of the paper. The cell sheet becomes a living body stimulated tissue through the following steps.
(S101): Cells are inoculated.
At this time, isolated stem cells 202 are floating in a culture medium.
(S102): Cells adhere to the culture surface 201. At this time, the density of stem cells 202 is sparse.
(S103): Cells proliferate on the entire culture surface in a monolayer to form a basal layer.
(S104): Cells are stratified in two or more layers. That is, the cells form a stratified structure. The cells in the second and higher layers differentiate to form a cell sheet. In the differentiation, proteins expressed in the cells are different depending on the layer.
(S105): The cell sheet is peeled off from the culture surface and transplanted to an affected part.
In the current circumstances, the quality of the cell sheet used for transplantation is verified by observation of the cell sheet under culture by the phase-contrast microscopy. Alternatively, it is verified by an invasive evaluation over a cell sheet for evaluation produced simultaneously on the same conditions as those for the cell sheet for transplantation, such as the tissue staining.
However, these methods have the following problems: The cell observation by the phase-contrast microscopy is non-invasive, and is performed at any time during cell culture. However, the observation can only be applied to the surface layer of cell sheet, and cannot evaluate the stratified cell sheet in step (S104) and later steps. Although the tissue staining evaluation currently performed on cell sheets for evaluation can evaluate the degree of stratification or differentiation, but it is an invasive technique for fixing the cell sheet and cannot evaluate a sheet for transplantation itself. It can be the that the establishment of a non-invasive measurement technique which solves these problems contributes to an improvement in the quality of regenerative tissues for transplantation by enabling evaluation of cell states of cell sheets for transplantation directly.
Non-invasive cell evaluation techniques have been described in some documents so far. For example, Patent Literature 1 describes a method in which an optical microscope is used to photograph a plurality of images with the focal points being different Z position and cells adhered to the culture surface and those which have peeled off are determined.